US20060121041A1

US20060121041A1 - Gpcs as modifiers of the irrtk p21 pathways and methods of use

Info

Publication number: US20060121041A1
Application number: US10/483,789
Authority: US
Inventors: Lori Friedman; Gregory Plowman; Tom Kidd; Roel Funke; Danxi Li; Siobhan Roche
Original assignee: Exelixis Inc
Current assignee: Exelixis Inc
Priority date: 2001-07-12
Filing date: 2002-07-10
Publication date: 2006-06-08
Also published as: EP1412477A4; CA2454135A1; WO2003006611A2; JP2005520485A; EP1412477A2; WO2003006611A3

Abstract

Human GPC genes are identified as modulators of the IRRTK or p21 pathways, and thus are therapeutic targets for disorders associated with defective IRRTK or p21 function. Methods for identifying modulators of IRRTK or p21, comprising screening for agents that modulate the activity of GPC are provided.

Description

REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. provisional patent applications 60/305,016 filed Jul. 12, 2001, and 60/328,507 filed Oct. 10, 2001. The contents of the prior applications are hereby incorporated in their entirety.

BACKGROUND OF THE INVENTION

Signal transduction pathways are made up of growth factors, their receptors, upstream regulators of the growth factors, and downstream intracellular kinase networks. These pathways regulate and play crucial roles in many cellular processes, such as metabolism and proliferation.
In humans, there are three members of the Insulin Receptor family of receptor tyrosine kinases (IRRTK): Insulin Receptor (InsR), Insulin like growth factor receptor (IGFR), and insulin receptor related receptor (IRR).
The insulin receptor (InR) binds insulin, the major anabolic hormone in humans, and through subsequent receptor and substrate phosphorylation, elicits a pleiotropic response, activating multiple signaling pathways for metabolic (carbohydrate, lipid, and protein processing) and growth (cell proliferation and differentiation) control (Smith R M, et al., Int Rev Cytol 1997; 173:243-80).
The type 1 insulin-like growth factor receptor (IGF-1R), a transmembrane tyrosine kinase, is widely expressed across many cell types in fetal and postnatal tissues, including motor and sensory neurons and glial cells. Activation of the receptor following binding of the secreted growth factor ligands IGF-1 and IGF-2 elicits a repertoire of cellular responses including proliferation, and the protection of cells from programmed cell death or apoptosis. As a result, signaling through the IGF-1R is the principal pathway responsible for somatic growth. Emerging evidence suggests that members of the IGF family, including IGF-1, IGF-2, the IGF-1 receptor (IGF-1R), and the IGF binding proteins (IGFBPs), also play important roles in the development and progression of cancer. Both in vitro and in vivo studies show that IGFs are strong mitogens for a variety of cancer cells. IGF-1 also has an antiapoptotic action on cancer. IGF-1R, overexpressed in cancer cells, mediates the effects of IGFs and plays a role in cell transformation induced by tumor virus and oncogene products (Yu H, Berkel H. J La State Med Soc April 1999;151(4):218-23). Accordingly, genes identified in the IGFR growth signaling pathway are of interest as targets for proliferation, apoptosis, and neuronal survival.
Insulin receptor-related receptor (IRR) is an orphan receptor in the insulin receptor (IR) family of receptor tyrosine kinases, and is primarily localized to neural crest-derived sensory neurons during embryonic development where it might play a key role in neuronal survival. In adults, it is also expressed in pancreatic beta cells. IRR has no known ligands (Hirayama I, et al., Diabetes June 1999;48(6):1237-44; Tsujimoto K, et al., Neurosci Lett. Mar. 24, 1995;188(2):105-8; Reinhardt R R, et al., J Neurosci. August 1994;14(8):4674-83; Reinhardt R R, et al., Endocrinology. July 1993;133(1):3-10).
In the Drosophila there is only one member of the IRRTKs, known as InR, thus subserving the function of its three human homologues. Interestingly, in Drosophila the insulin receptor can drive proliferation if expressed in undifferentiated tissue.
The p21/CDKN1/WAF1/CIP1 protein(El-Deiry, W. S.; et al. Cell 75: 817-825, 1993; Harper, J. W.; et al. Cell 75: 805-816, 1993; Huppi, Ket al. Oncogene 9: 3017-3020, 1994) is a cell cycle control protein that inhibits cyclin-kinase activity, is tightly regulated at the transcriptional level by p21, and mediates p21 suppression of tumor cell growth. Along with p21, p21 appears to be essential for maintaining the G2 checkpoint in human cells (Bunz, F.; Dutriaux, A.; et al. Science 282:1497-1501, 1998). Sequences of P21 are well-conserved throughout evolution, and have been identified in species as diverse as human (Genbank Identifier 13643057), Drosophila melanogaster (GI#1684911), Caenorhabditis elegans (GI#4966283), and yeast (GI#2656016).
Glypicans (GPC) are proteins with very characteristic structures that are substituted with heparan sulfate and that are linked to the cell surface via glycosylphosphatidylinositol. The modular structure of the glypicans has been highly conserved throughout evolution. Six glypicans have been identified so far in vertebrates. Mutations in Drosophila, humans and mice reveal a role for these cell surface molecules in the control of cell growth, migration, and differentiation (De Cat B, and David G. Semin Cell Dev Biol April 2001;12(2):117-25; Higashiyama Set al (1993) J Cell Biol. 122:933-940).
Many GPCs play important roles in various disease conditions. GPC1 regulates growth factor action in pancreatic carcinoma cells and is overexpressed in human pancreatic cancer, and its expression in this cancer may enhance tumorigenic potential in vivo (Kleeff, J., et al. (1999) Pancreas 19:281-8; Kleeff, J., et al. (1998) J Clin Invest 102:1662-73; WO200023109). Levels of GPC1 are also increased in breast cacer (WO200023109). GPC3 is expressed in hepatocarcinoma and hepatoma, and is downregulated in mesothelioma primary tumor (Hsu, H. C., et al. (1997) Cancer Res 57:5179-84; Murthy, S. S., et al. (2000) Oncogene 19:410-6). Mutations in GPC3 are associated with Simpson-Golabi-Behmel syndrome (Pilia, G., et al. (1996) Nat Genet 12, 241-7). GPC4 is expressed in various cell lines including mammary gland and breast tumor cell lines, and variant forms of GPC4 coexisting with GPC3 variant forms may explain phenotypic variations in individuals displaying Simpson-Golabi-Behmel syndrome (Veugelers, M., et al. (1998) Genomics 53:1-11). GPC4 may be involved in diseases of abnormal cell growth and behavior (WO99/37764).
The ability to manipulate the genomes of model organisms such as Drosophila provides a powerful means to analyze biochemical processes that, due to significant evolutionary conservation, have direct relevance to more complex vertebrate organisms. Due to a high level of gene and pathway conservation, the strong similarity of cellular processes, and the functional conservation of genes between these model organisms and mammals, identification of the involvement of novel genes in particular pathways and their functions in such model organisms can directly contribute to the understanding of the correlative pathways and methods of modulating them in mammals (see, for example, Mechler B M et al., 1985 EMBO J 4:1551-1557; Gateff E. 1982 Adv. Cancer Res. 37: 33-74; Watson K L., et al., 1994 J Cell Sci. 18: 19-33; Miklos G L, and Rubin G M. 1996 Cell 86:521-529; Wassarman D A, et al., 1995 Curr Opin Gen Dev 5: 44-50; and Booth D R. 1999 Cancer Metastasis Rev. 18: 261-284). For example, a genetic screen can be carried out in an invertebrate model organism having underexpression (e.g. knockout) or overexpression of a gene (referred to as a “genetic entry point”) that yields a visible phenotype. Additional genes are mutated in a random or targeted manner. When a gene mutation changes the original phenotype caused by the mutation in the genetic entry point, the gene is identified as a “modifier” involved in the same or overlapping pathway as the genetic entry point. When the genetic entry point is an ortholog of a human gene implicated in a disease pathway, such as IRRTK or p21, modifier genes can be identified that may be attractive candidate targets for novel therapeutics.
All references cited herein, including sequence information in referenced Genbank identifier numbers and website references, are incorporated herein in their entireties.

SUMMARY OF THE INVENTION

We have discovered genes that modify the IRRTK and p21 pathways in and Drosophila, and identified their human orthologs, hereinafter referred to as GPC. The invention provides methods for utilizing these IRRTK or p21 modifier genes and polypeptides to identify GPC-modulating agents that are candidate therapeutic agents that can be used in the treatment of disorders associated with defective or impaired IRRTK or p21 function and/or GPC function. Preferred GPC-modulating agents specifically bind to GPC polypeptides and restore IRRTK or p21 function. Other preferred GPC-modulating agents are nucleic acid modulators such as antisense oligomers and RNAi that repress GPC gene expression or product activity by, for example, binding to and inhibiting the respective nucleic acid (i.e. DNA or mRNA).
GPC modulating agents may be evaluated by any convenient in vitro or in vivo assay for molecular interaction with a GPC polypeptide or nucleic acid. In one embodiment, candidate GPC modulating agents are tested with an assay system comprising a GPC polypeptide or nucleic acid. Agents that produce a change in the activity of the assay system relative to controls are identified as candidate IRRTK or p21 modulating agents. The assay system may be cell-based or cell-free. GPC-modulating agents include GPC related proteins (e.g. dominant negative mutants, and biotherapeutics); GPC-specific antibodies; GPC-specific antisense oligomers and other nucleic acid modulators; and chemical agents that specifically bind to or interact with GPC or compete with GPC binding partner (e.g. by binding to a GPC binding partner). In one specific embodiment, a small molecule modulator is identified using a binding assay. In specific embodiments, the screening assay system is selected from an apoptosis assay, a cell proliferation assay, an angiogenesis assay, and a hypoxic induction assay.
In another embodiment, candidate IRRTK or p21 pathways modulating agents are further tested using a second assay system that detects changes in the IRRTK or p21 pathways, such as angiogenic, apoptotic, or cell proliferation changes produced by the originally identified candidate agent or an agent derived from the original agent. The second assay system may use cultured cells or non-human animals. In specific embodiments, the secondary assay system uses non-human animals, including animals predetermined to have a disease or disorder implicating the IRRTK or p21 pathways, such as an angiogenic, apoptotic, or cell proliferation disorder (e.g. cancer).
The invention further provides methods for modulating the GPC function and/or the IRRTK or p21 pathways in a mammalian cell by contacting the mammalian cell with an agent that specifically binds a GPC polypeptide or nucleic acid. The agent may be a small molecule modulator, a nucleic acid modulator, or an antibody and may be administered to a mammalian animal predetermined to have a pathology associated the IRRTK or p21 pathways.

DETAILED DESCRIPTION OF THE INVENTION

Genetic screens were designed to identify modifiers of the IRRTK or p21 pathways in Drosophila. For IRRTK, the dominant negative form of InR was expressed in the eye, resulting in a small eye phenotype. For p21, human p21 gene was overexpressed in the eye, resulting in a small, rough eye phenotype. Both screens aimed to identify enhancers or suppressors of the eye phenotype. The Dally gene was identified as a modifier of both IRRTK and p21 pathways. Accordingly, vertebrate orthologs of these modifiers, and preferably the human orthologs, glypican (GPC) genes (i.e., nucleic acids and polypeptides) are attractive drug targets for the treatment of pathologies associated with a defective IRRTK or p21 signaling pathway, such as cancer.
In vitro and in vivo methods of assessing GPC function are provided herein. Modulation of the GPC or their respective binding partners is useful for understanding the association of the IRRTK or p21 pathways and its members in normal and disease conditions and for developing diagnostics and therapeutic modalities for IRRTK or p21 related pathologies. GPC-modulating agents that act by inhibiting or enhancing GPC expression, directly or indirectly, for example, by affecting a GPC function such as binding activity, can be identified using methods provided herein. GPC modulating agents are useful in diagnosis, therapy and pharmaceutical development.
Nucleic Acids and Polypeptides of the Invention
Sequences related to GPC nucleic acids and polypeptides that can be used in the invention are disclosed in Genbank (referenced by Genbank identifier (GI) number) as GI#s 4504080 (SEQ ID NO: 1), 18567116 (SEQ ID NO:3), 13632290 (SEQ ID NO:4), 13632295 (SEQ ID NO:5), 4504082 (SEQ ID NO:6), 5360214 (SEQ ID NO:8), 3015541 (SEQ ID NO:9), 4877642 (SEQ ID NO: 10), and 8051601 (SEQ ID NO: 11) for nucleic acid, and GI#s 4504081 (SEQ ID NO:13), 1708021 (SEQ ID NO:14), 13632291 (SEQ ID NO:16), 4758462 (SEQ ID NO:17), 11421168 (SEQ ID NO:18), 4504083 (SEQ ID NO:19), 4758464 (SEQ ID NO:20), 9973298 (SEQ ID NO:21) and 5031719 (SEQ ID NO:22) for polypeptides. Additionally, nucleic acid sequences of SEQ ID NOs: 2, 7, 12, 23 and 24 amino acid sequence of SEQ ID NO: 15 can be used in the invention.
GPCs are glycosylphosphatidylinositol-anchored cell surface heparan sulfate proteoglycan proteins with glypican domains. The term “GPC polypeptide” refers to a full-length GPC protein or a functionally active fragment or derivative thereof. A “functionally active” GPC fragment or derivative exhibits one or more functional activities associated with a full-length, wild-type GPC protein, such as antigenic or immunogenic activity, ability to bind natural cellular substrates, etc. The functional activity of GPC proteins, derivatives and fragments can be assayed by various methods known to one skilled in the art (Current Protocols in Protein Science (1998) Coligan et al., eds., John Wiley & Sons, Inc., Somerset, N.J.) and as further discussed below. For purposes herein, functionally active fragments also include those fragments that comprise one or more structural domains of a GPC, such as a binding domain. Protein domains can be identified using the PFAM program (Bateman A., et al., Nucleic Acids Res, 1999, 27:260-2). For example, the glypican domains (PFAM 01153) of GPC from GI#s4504081, 4758462, 4504083, 4758464, and 5031719 (SEQ ID NOs:13, 17, 19, 20, and 22, respectively) are located at approximately amino acid residues 2 to 557, 4 to 578, 1 to 555, 2 to 572, and 7 to 554, respectively. Methods for obtaining GPC polypeptides are also further described below. In some embodiments, preferred fragments are functionally active, domain-containing fragments comprising at least 25 contiguous amino acids, preferably at least 50, more preferably 75, and most preferably at least 100 contiguous amino acids of any one of SEQ ID NOs:13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 (a GPC). In further preferred embodiments, the fragment comprises the entire glypican (functionally active) domain.
The term “GPC nucleic acid” refers to a DNA or RNA molecule that encodes a GPC polypeptide. Preferably, the GPC polypeptide or nucleic acid or fragment thereof is from a human, but can also be an ortholog, or derivative thereof with at least 70% sequence identity, preferably at least 80%, more preferably 85%, still more preferably 90%, and most preferably at least 95% sequence identity with GPC. Normally, orthologs in different species retain the same function, due to presence of one or more protein motifs and/or 3-dimensional structures. Orthologs are generally identified by sequence homology analysis, such as BLAST analysis, usually using protein bait sequences. Sequences are assigned as a potential ortholog if the best hit sequence from the forward BLAST result retrieves the original query sequence in the reverse BLAST (Huynen M A and Bork P, Proc Natl Acad Sci (1998) 95:5849-5856; Huynen M A et al., Genome Research (2000) 10: 1204-1210). Programs for multiple sequence alignment, such as CLUSTAL (Thompson J D et al, 1994, Nucleic Acids Res 22:4673-4680) may be used to highlight conserved regions and/or residues of orthologous proteins and to generate phylogenetic trees. In a phylogenetic tree representing multiple homologous sequences from diverse species (e.g., retrieved through BLAST analysis), orthologous sequences from two species generally appear closest on the tree with respect to all other sequences from these two species. Structural threading or other analysis of protein folding (e.g., using software by ProCeryon, Biosciences, Salzburg, Austria) may also identify potential orthologs. In evolution, when a gene duplication event follows speciation, a single gene in one species, such as Drosophila, may correspond to multiple genes (paralogs) in another, such as human. As used herein, the term “orthologs” encompasses paralogs. As used herein, “percent (%) sequence identity” with respect to a subject sequence, or a specified portion of a subject sequence, is defined as the percentage of nucleotides or amino acids in the candidate derivative sequence identical with the nucleotides or amino acids in the subject sequence (or specified portion thereof), after aligning the sequences and introducing gaps, if necessary to achieve the maximum percent sequence identity, as generated by the program WU-BLAST-2.0a19 (Altschul et al., J. Mol. Biol. (1997) 215:403-410) with all the search parameters set to default values. The HSP S and HSP S2 parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched. A % identity value is determined by the number of matching identical nucleotides or amino acids divided by the sequence length for which the percent identity is being reported. “Percent (%) amino acid sequence similarity” is determined by doing the same calculation as for determining % amino acid sequence identity, but including conservative amino acid substitutions in addition to identical amino acids in the computation.
A conservative amino acid substitution is one in which an amino acid is substituted for another amino acid having similar properties such that the folding or activity of the protein is not significantly affected. Aromatic amino acids that can be substituted for each other are phenylalanine, tryptophan, and tyrosine; interchangeable hydrophobic amino acids are leucine, isoleucine, methionine, and valine; interchangeable polar amino acids are glutamine and asparagine; interchangeable basic amino acids are arginine, lysine and histidine; interchangeable acidic amino acids are aspartic acid and glutamic acid; and interchangeable small amino acids are alanine, serine, threonine, cysteine and glycine.
Alternatively, an alignment for nucleic acid sequences is provided by the local homology algorithm of Smith and Waterman (Smith and Waterman, 1981, Advances in Applied Mathematics 2:482-489; database: European Bioinformatics Institute; Smith and Waterman, 1981, J. of Molec. Biol., 147:195-197; Nicholas et al., 1998, “A Tutorial on Searching Sequence Databases and Sequence Scoring Methods” (www.psc.edu) and references cited therein.; W. R. Pearson, 1991, Genomics 11:635-650). This algorithm can be applied to amino acid sequences by using the scoring matrix developed by Dayhoff (Dayhoff: Atlas of Protein Sequences and Structure, M. O. Dayhoff ed., 5 suppl. 3:353-358, National Biomedical Research Foundation, Washington, D.C., USA), and normalized by Gribskov (Gribskov 1986 Nucl. Acids Res. 14(6):6745-6763). The Smith-Waterman algorithm may be employed where default parameters are used for scoring (for example, gap open penalty of 12, gap extension penalty of two). From the data generated, the “Match” value reflects “sequence identity.”
Derivative nucleic acid molecules of the subject nucleic acid molecules include sequences that hybridize to the nucleic acid sequence of any of SEQ ID NOs:1, 2, 3, 4, 5, 6, 7 ,8 ,9 10, 11, 12, 23 or 24. The stringency of hybridization can be controlled by temperature, ionic strength, pH, and the presence of denaturing agents such as formamide during hybridization and washing. Conditions routinely used are set out in readily available procedure texts (e.g., Current Protocol in Molecular Biology, Vol. 1, Chap. 2.10, John Wiley & Sons, Publishers (1994); Sambrook et al., Molecular Cloning, Cold Spring Harbor (1989)). In some embodiments, a nucleic acid molecule of the invention is capable of hybridizing to a nucleic acid molecule containing the nucleotide sequence of any one of SEQ ID NOs:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 under stringent hybridization conditions that comprise: prehybridization of filters containing nucleic acid for 8 hours to overnight at 65° C. in a solution comprising 6× single strength citrate (SSC) (1×SSC is 0.15 M NaCl, 0.015 M Na citrate; pH 7.0), 5× Denhardt's solution, 0.05% sodium pyrophosphate and 100 μg/ml herring sperm DNA; hybridization for 18-20 hours at 65° C. in a solution containing 6×SSC, 1× Denhardt's solution, 100 μg/ml yeast tRNA and 0.05% sodium pyrophosphate; and washing of filters at 65° C. for 1 h in a solution containing 0.2×SSC and 0.1% SDS (sodium dodecyl sulfate).
In other embodiments, moderately stringent hybridization conditions are used that comprise: pretreatment of filters containing nucleic acid for 6 h at 40° C. in a solution containing 35% formamide, 5×SSC, 50 mM Tris-HCl (pH7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 μg/ml denatured salmon sperm DNA; hybridization for 18-20 h at 40° C. in a solution containing 35% formamide, 5×SSC, 50 mM Tris-HCl (pH7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg/ml salmon sperm DNA, and 10% (wt/vol) dextran sulfate; followed by washing twice for 1 hour at 55° C. in a solution containing 2×SSC and 0.1% SDS.
Alternatively, low stringency conditions can be used that comprise: incubation for 8 hours to overnight at 37° C. in a solution comprising 20% formamide, 5×SSC, 50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured sheared salmon sperm DNA; hybridization in the same buffer for 18 to 20 hours; and washing of filters in 1×SSC at about 37° C. for 1 hour.
Isolation, Production, Expression, and Mis-Expression of GPC Nucleic Acids and Polypeptides
GPC nucleic acids and polypeptides, useful for identifying and testing agents that modulate GPC function and for other applications related to the involvement of GPC in the IRRTK or p21 pathways. GPC nucleic acids and derivatives and orthologs thereof may be obtained using any available method. For instance, techniques for isolating cDNA or genomic DNA sequences of interest by screening DNA libraries or by using polymerase chain reaction (PCR) are well known in the art. In general, the particular use for the protein will dictate the particulars of expression, production, and purification methods. For instance, production of proteins for use in screening for modulating agents may require methods that preserve specific biological activities of these proteins, whereas production of proteins for antibody generation may require structural integrity of particular epitopes. Expression of proteins to be purified for screening or antibody production may require the addition of specific tags (e.g., generation of fusion proteins). Overexpression of a GPC protein for assays used to assess GPC function, such as involvement in cell cycle regulation or hypoxic response, may require expression in eukaryotic cell lines capable of these cellular activities. Techniques for the expression, production, and purification of proteins are well known in the art; any suitable means therefore may be used (e.g., Higgins S J and Hames B D (eds.) Protein Expression: A Practical Approach, Oxford University Press Inc., New York 1999; Stanbury P F et al., Principles of Fermentation Technology, 2^ndedition, Elsevier Science, New York, 1995; Doonan S (ed.) Protein Purification Protocols, Humana Press, New Jersey, 1996; Coligan J E et al, Current Protocols in Protein Science (eds.), 1999, John Wiley & Sons, New York). In particular embodiments, recombinant GPC is expressed in a cell line known to have defective p21 function such as HCT116 colon cancer cells available from American Type Culture Collection (ATCC), Manassas, Va.). The recombinant cells are used in cell-based screening assay systems of the invention, as described further below.
The nucleotide sequence encoding a GPC polypeptide can be inserted into any appropriate expression vector. The necessary transcriptional and translational signals, including promoter/enhancer element, can derive from the native GPC gene and/or its flanking regions or can be heterologous. A variety of host-vector expression systems may be utilized, such as mammalian cell systems infected with virus (e.g. vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g. baculovirus); microorganisms such as yeast containing yeast vectors, or bacteria transformed with bacteriophage, plasmid, or cosmid DNA. A host cell strain that modulates the expression of, modifies, and/or specifically processes the gene product may be used.
To detect expression of the GPC gene product, the expression vector can comprise a promoter operably linked to a GPC gene nucleic acid, one or more origins of replication, and, one or more selectable markers (e.g. thymidine kinase activity, resistance to antibiotics, etc.). Alternatively, recombinant expression vectors can be identified by assaying for the expression of the GPC gene product based on the physical or functional properties of the GPC protein in in vitro assay systems (e.g. immunoassays).
The GPC protein, fragment, or derivative may be optionally expressed as a fusion, or chimeric protein product (i.e. it is joined via a peptide bond to a heterologous protein sequence of a different protein), for example to facilitate purification or detection. A chimeric product can be made by ligating the appropriate nucleic acid sequences encoding the desired amino acid sequences to each other using standard methods and expressing the chimeric product. A chimeric product may also be made by protein synthetic techniques, e.g. by use of a peptide synthesizer (Hunkapiller et al., Nature (1984) 310:105-111).
Once a recombinant cell that expresses the GPC gene sequence is identified, the gene product can be isolated and purified using standard methods (e.g. ion exchange, affinity, and gel exclusion chromatography; centrifugation; differential solubility; electrophoresis). Alternatively, native GPC proteins can be purified from natural sources, by standard methods (e.g. immunoaffinity purification). Once a protein is obtained, it may be quantified and its activity measured by appropriate methods, such as immunoassay, bioassay, or other measurements of physical properties, such as crystallography.
The methods of this invention may also use cells that have been engineered for altered expression (mis-expression) of GPC or other genes associated with the IRRTK or p21 pathways. As used herein, mis-expression encompasses ectopic expression, over-expression, under-expression, and non-expression (e.g. by gene knock-out or blocking expression that would otherwise normally occur).
Genetically Modified Animals
Animal models that have been genetically modified to alter GPC expression may be used in in vivo assays to test for activity of a candidate IRRTK or p21 modulating agent, or to further assess the role of GPC in a IRRTK or p21 pathways process such as apoptosis or cell proliferation. Preferably, the altered GPC expression results in a detectable phenotype, such as decreased or increased levels of cell proliferation, angiogenesis, or apoptosis compared to control animals having normal GPC expression. The genetically modified animal may additionally have altered IRRTK or p21 expression (e.g. IRRTK or p21 knockout). Preferred genetically modified animals are mammals such as primates, rodents (preferably mice), cows, horses, goats, sheep, pigs, dogs and cats. Preferred non-mammalian species include zebrafish, C. elegans, and Drosophila. Preferred genetically modified animals are transgenic animals having a heterologous nucleic acid sequence present as an extrachromosomal element in a portion of its cells, i.e. mosaic animals (see, for example, techniques described by Jakobovits, 1994, Curr. Biol. 4:761-763.) or stably integrated into its germ line DNA (i.e., in the genomic sequence of most or all of its cells). Heterologous nucleic acid is introduced into the germ line of such transgenic animals by genetic manipulation of, for example, embryos or embryonic stem cells of the host animal.
Methods of making transgenic animals are well-known in the art (for transgenic mice see Brinster et al., Proc. Nat. Acad. Sci. USA 82: 4438-4442 (1985), U.S. Pat. Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No. 4,873,191 by Wagner et al., and Hogan, B., Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1986); for particle bombardment see U.S. Pat. No., 4,945,050, by Sandford et al.; for transgenic Drosophila see Rubin and Spradling, Science (1982) 218:348-53 and U.S. Pat. No. 4,670,388; for transgenic insects see Berghammer A. J. et al., A Universal Marker for Transgenic Insects (1999) Nature 402:370-371; for transgenic Zebrafish see Lin S., Transgenic Zebrafish, Methods Mol Biol. (2000);136:375-3830); for microinjection procedures for fish, amphibian eggs and birds see Houdebine and Chourrout, Experientia (1991) 47:897-905; for transgenic rats see Hammer et al., Cell (1990) 63:1099-1112; and for culturing of embryonic stem (ES) cells and the subsequent production of transgenic animals by the introduction of DNA into ES cells using methods such as electroporation, calcium phosphate/DNA precipitation and direct injection see, e.g., Teratocarcinomas and Embryonic Stem Cells, A Practical Approach, E. J. Robertson, ed., IRL Press (1987)). Clones of the nonhuman transgenic animals can be produced according to available methods (see Wilmut, I. et al. (1997) Nature 385:810-813; and PCT International Publication Nos. WO 97/07668 and WO 97/07669).
In one embodiment, the transgenic animal is a “knock-out” animal having a heterozygous or homozygous alteration in the sequence of an endogenous GPC gene that results in a decrease of GPC function, preferably such that GPC expression is undetectable or insignificant. Knock-out animals are typically generated by homologous recombination with a vector comprising a transgene having at least a portion of the gene to be knocked out. Typically a deletion, addition or substitution has been introduced into the transgene to functionally disrupt it. The transgene can be a human gene (e.g., from a human genomic clone) but more preferably is an ortholog of the human gene derived from the transgenic host species. For example, a mouse GPC gene is used to construct a homologous recombination vector suitable for altering an endogenous GPC gene in the mouse genome. Detailed methodologies for homologous recombination in mice are available (see Capecchi, Science (1989) 244:1288-1292; Joyner et al., Nature (1989) 338:153-156). Procedures for the production of non-rodent transgenic mammals and other animals are also available (Houdebine and Chourrout, supra; Pursel et al., Science (1989) 244:1281-1288; Simms etal., Bio/Technology (1988) 6:179-183). In apreferred embodiment, knock-out animals, such as mice harboring a knockout of a specific gene, may be used to produce antibodies against the human counterpart of the gene that has been knocked out (Claesson M H et al., (1994) Scan J Immunol 40:257-264; Declerck P J et al., (1995) J Biol Chem. 270:8397-400).
In another embodiment, the transgenic animal is a “knock-in” animal having an alteration in its genome that results in altered expression (e.g., increased (including ectopic) or decreased expression) of the GPC gene, e.g., by introduction of additional copies of GPC, or by operatively inserting a regulatory sequence that provides for altered expression of an endogenous copy of the GPC gene. Such regulatory sequences include inducible, tissue-specific, and constitutive promoters and enhancer elements. The knock-in can be homozygous or heterozygous.
Transgenic nonhuman animals can also be produced that contain selected systems allowing for regulated expression of the transgene. One example of such a system that may be produced is the cre/loxP recombinase system of bacteriophage P1 (Lakso et al., PNAS (1992) 89:6232-6236; U.S. Pat. No. 4,959,317). If a cre/loxP recombinase system is used to regulate expression of the transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein are required. Such animals can be provided through the construction of “double” transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase. Another example of a recombinase system is the FLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al. (1991) Science 251:1351-1355; U.S. Pat. No. 5,654,182). In a preferred embodiment, both Cre-LoxP and Flp-Frt are used in the same system to regulate expression of the transgene, and for sequential deletion of vector sequences in the same cell (Sun X et al (2000) Nat Genet 25:83-6).
The genetically modified animals can be used in genetic studies to further elucidate the IRRTK or p21 pathways, as animal models of disease and disorders implicating defective IRRTK or p21 function, and for in vivo testing of candidate therapeutic agents, such as those identified in screens described below. The candidate therapeutic agents are administered to a genetically modified animal having altered GPC function and phenotypic changes are compared with appropriate control animals such as genetically modified animals that receive placebo treatment, and/or animals with unaltered GPC expression that receive candidate therapeutic agent.
In addition to the above-described genetically modified animals having altered GPC function, animal models having defective IRRTK or p21 function (and otherwise normal GPC function), can be used in the methods of the present invention. For example, a IRRTK or p21 knockout mouse can be used to assess, in vivo, the activity of a candidate IRRTK or p21 modulating agent identified in one of the in vitro assays described below. p21 knockout mice are described in the literature (Umanoff H, et al., Proc Natl Acad Sci USA Feb. 28, 1995;92(5): 1709-13). Preferably, the candidate IRRTK or p21 modulating agent when administered to a model system with cells defective in IRRTK or p21 function, produces a detectable phenotypic change in the model system indicating that the IRRTK or p21 function is restored, i.e., the cells exhibit normal cell cycle progression.
Modulating Agents
The invention provides methods to identify agents that interact with and/or modulate the function of GPC and/or the IRRTK or p21 pathways. Modulating agents identified by the methods are also part of the invention. Such agents are useful in a variety of diagnostic and therapeutic applications associated with the IRRTK or p21 pathways, as well as in further analysis of the GPC protein and its contribution to the IRRTK or p21 pathways. Accordingly, the invention also provides methods for modulating the IRRTK or p21 pathways comprising the step of specifically modulating GPC activity by administering a GPC-interacting or -modulating agent.
As used herein, an “GPC-modulating agent” is any agent that modulated GPC function, for example, an agent that interacts with GPC to inhibit or enhance GPC activity or otherwise affect normal GPC function. GPC function can be affected at any level, including transcription, protein expression, protein localization, and cellular or extra-cellular activity. In a preferred embodiment, the GPC-modulating agent specifically modulates the function of the GPC. The phrases “specific modulating agent”, “specifically modulates”, etc., are used herein to refer to modulating agents that directly bind to the GPC polypeptide or nucleic acid, and preferably inhibit, enhance, or otherwise alter, the function of the GPC. These phrases also encompasses modulating agents that alter the interaction of the GPC with a binding partner, substrate, or cofactor (e.g. by binding to a binding partner of a GPC, or to a protein/binding partner complex, and altering GPC function). In a further preferred embodiment, the GPC-modulating agent is a modulator of the IRRTK or p21 pathways (e.g. it restores and/or upregulates IRRTK or p21 function) and thus is also a IRRTK or p21-modulating agent.
Preferred GPC-modulating agents include small molecule compounds; GPC-interacting proteins, including antibodies and other biotherapeutics; and nucleic acid modulators such as antisense and RNA inhibitors. The modulating agents may be formulated in pharmaceutical compositions, for example, as compositions that may comprise other active ingredients, as in combination therapy, and/or suitable carriers or excipients. Techniques for formulation and administration of the compounds may be found in “Remington's Pharmaceutical Sciences” Mack Publishing Co., Easton, Pa., 19^thedition.
Small Molecule Modulators
Small molecules, are often preferred to modulate function of proteins with enzymatic function, and/or containing protein interaction domains. Chemical agents, referred to in the art as “small molecule” compounds are typically organic, non-peptide molecules, having a molecular weight less than 10,000, preferably less than 5,000, more preferably less than 1,000, and most preferably less than 500. This class of modulators includes chemically synthesized molecules, for instance, compounds from combinatorial chemical libraries. Synthetic compounds may be rationally designed or identified based on known or inferred properties of the GPC protein or may be identified by screening compound libraries. Alternative appropriate modulators of this class are natural products, particularly secondary metabolites from organisms such as plants or fungi, which can also be identified by screening compound libraries for GPC-modulating activity. Methods for generating and obtaining compounds are well known in the art (Schreiber S L, Science (2000)151:1964-1969; Radmann J and Gunther J, Science (2000) 151:1947-1948).
Small molecule modulators identified from screening assays, as described below, can be used as lead compounds from which candidate clinical compounds may be designed, optimized, and synthesized. Such clinical compounds may have utility in treating pathologies associated with the IRRTK or p21 pathways. The activity of candidate small molecule modulating agents may be improved several-fold through iterative secondary functional validation, as further described below, structure determination, and candidate modulator modification and testing. Additionally, candidate clinical compounds are generated with specific regard to clinical and pharmacological properties. For example, the reagents may be derivatized and re-screened using in vitro and in vivo assays to optimize activity and minimize toxicity for pharmaceutical development.
Protein Modulators
Specific GPC-interacting proteins are useful in a variety of diagnostic and therapeutic applications related to the IRRTK or p21 pathways and related disorders, as well as in validation assays for other GPC-modulating agents. In a preferred embodiment, GPC-interacting proteins affect normal GPC function, including transcription, protein expression, protein localization, and cellular or extra-cellular activity. In another embodiment, GPC-interacting proteins are useful in detecting and providing information about the function of GPC proteins, as is relevant to IRRTK or p21 related disorders, such as cancer (e.g., for diagnostic means).
An GPC-interacting protein may be endogenous, i.e. one that naturally interacts genetically or biochemically with a GPC, such as a member of the GPC pathway that modulates GPC expression, localization, and/or activity. GPC-modulators include dominant negative forms of GPC-interacting proteins and of GPC proteins themselves. Yeast two-hybrid and variant screens offer preferred methods for identifying endogenous GPC-interacting proteins (Finley, R. L. et al. (1996) in DNA Cloning-Expression Systems: A Practical Approach, eds. Glover D. & Hames B. D (Oxford University Press, Oxford, England), pp. 169-203; Fashema S F et al., Gene (2000) 250:1-14; Drees B L Curr Opin Chem Biol (1999) 3:64-70; Vidal M and Legrain P Nucleic Acids Res (1999) 27:919-29; and U.S. Pat. No. 5,928,868). Mass spectrometry is an alternative preferred method for the elucidation of protein complexes (reviewed in, e.g., Pandley A and Mann M, Nature (2000) 405:837-846; Yates J R 3^rd, Trends Genet (2000) 16:5-8).
An GPC-interacting protein may be an exogenous protein, such as a GPC-specific antibody or a T-cell antigen receptor (see, e.g., Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory; Harlow and Lane (1999) Using antibodies: a laboratory manual. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press). GPC antibodies are further discussed below.
In preferred embodiments, a GPC-interacting protein specifically binds a GPC protein. In alternative preferred embodiments, a GPC-modulating agent binds a GPC substrate, binding partner, or cofactor.
Antibodies
In another embodiment, the protein modulator is a GPC specific antibody agonist or antagonist. The antibodies have therapeutic and diagnostic utilities, and can be used in screening assays to identify GPC modulators. The antibodies can also be used in dissecting the portions of the GPC pathway responsible for various cellular responses and in the general processing and maturation of the GPC.
Antibodies that specifically bind GPC polypeptides can be generated using known methods. Preferably the antibody is specific to a mammalian ortholog of GPC polypeptide, and more preferably, to human GPC. Antibodies may be polyclonal, monoclonal (mAbs), humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab′).sub.2 fragments, fragments produced by a FAb expression library, anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above. Epitopes of GPC which are particularly antigenic can be selected, for example, by routine screening of GPC polypeptides for antigenicity or by applying a theoretical method for selecting antigenic regions of a protein (Hopp and Wood (1981), Proc. Nati. Acad. Sci. U.S.A. 78:3824-28; Hopp and Wood, (1983) Mol. Immunol. 20:483-89; Sutcliffe et al., (1983) Science 219:660-66) to the amino acid sequence shown in any of SEQ ID NOs:13, 14, 15, 16, 17, 18, 19, 20, 21, or 22. Monoclonal antibodies with affinities of 10⁸M⁻¹preferably 10⁹M⁻¹to 10¹⁰M⁻¹, or stronger can be made by standard procedures as described (Harlow and Lane, supra; Goding (1986) Monoclonal Antibodies: Principles and Practice (2d ed) Academic Press, New York; and U.S. Pat. Nos. 4,381,292; 4,451,570; and 4,618,577). Antibodies may be generated against crude cell extracts of GPC or substantially purified fragments thereof. If GPC fragments are used, they preferably comprise at least 10, and more preferably, at least 20 contiguous amino acids of a GPC protein. In a particular embodiment, GPC-specific antigens and/or immunogens are coupled to carrier proteins that stimulate the immune response. For example, the subject polypeptides are covalently coupled to the keyhole limpet hemocyanin (KLH) carrier, and the conjugate is emulsified in Freund's complete adjuvant, which enhances the immune response. An appropriate immune system such as a laboratory rabbit or mouse is immunized according to conventional protocols.
The presence of GPC-specific antibodies is assayed by an appropriate assay such as a solid phase enzyme-linked immunosorbant assay (ELISA) using immobilized corresponding GPC polypeptides. Other assays, such as radioimmunoassays or fluorescent assays might also be used.
Chimeric antibodies specific to GPC polypeptides can be made that contain different portions from different animal species. For instance, a human immunoglobulin constant region may be linked to a variable region of a murine mAb, such that the antibody derives its biological activity from the human antibody, and its binding specificity from the murine fragment. Chimeric antibodies are produced by splicing together genes that encode the appropriate regions from each species (Morrison et al., Proc. Natl. Acad. Sci. (1984) 81:6851-6855; Neuberger et al., Nature (1984) 312:604-608; Takeda et al., Nature (1985) 31:452-454). Humanized antibodies, which are a form of chimeric antibodies, can be generated by grafting complementary-determining regions (CDRs) (Carlos, T. M., J. M. Harlan. 1994. Blood 84:2068-2101) of mouse antibodies into a background of human framework regions and constant regions by recombinant DNA technology (Riechmann L M, et al., 1988 Nature 323: 323-327). Humanized antibodies contain ˜10% murine sequences and ˜90% human sequences, and thus further reduce or eliminate immunogenicity, while retaining the antibody specificities (Co M S, and Queen C. 1991 Nature 351: 501-501; Morrison S L. 1992 Ann. Rev. Immun. 10:239-265). Humanized antibodies and methods of their production are well-known in the art (U.S. Pat. Nos. 5,530,101, 5,585,089, 5,693,762, and 6,180,370).
GPC-specific single chain antibodies which are recombinant, single chain polypeptides formed by linking the heavy and light chain fragments of the Fv regions via an amino acid bridge, can be produced by methods known in the art (U.S. Pat. No. 4,946,778; Bird, Science (1988) 242:423-426; Huston et al., Proc. Nad. Acad. Sci. USA (1988) 85:5879-5883; and Ward et al., Nature (1989) 334:544-546).
Other suitable techniques for antibody production involve in vitro exposure of lymphocytes to the antigenic polypeptides or alternatively to selection of libraries of antibodies in phage or similar vectors (Huse et al., Science (1989) 246:1275-1281). As used herein, T-cell antigen receptors are included within the scope of antibody modulators (Harlow and Lane, 1988, supra).
The polypeptides and antibodies of the present invention may be used with or without modification. Frequently, antibodies will be labeled by joining, either covalently or non-covalently, a substance that provides for a detectable signal, or that is toxic to cells that express the targeted protein (Menard S, et al., Int J. Biol Markers (1989) 4:131-134). A wide variety of labels and conjugation techniques are known and are reported extensively in both the scientific and patent literature. Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, fluorescent emitting lanthanide metals, chemiluminescent moieties, bioluminescent moieties, magnetic particles, and the like (U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241). Also, recombinant immunoglobulins may be produced (U.S. Pat. No. 4,816,567). Antibodies to cytoplasmic polypeptides may be delivered and reach their targets by conjugation with membrane-penetrating toxin proteins (U.S. Pat. No. 6,086,900).
When used therapeutically in a patient, the antibodies of the subject invention are typically administered parenterally, when possible at the target site, or intravenously. The therapeutically effective dose and dosage regimen is determined by clinical studies. Typically, the amount of antibody administered is in the range of about 0.1 mg/kg-to about 10 mg/kg of patient weight. For parenteral administration, the antibodies are formulated in a unit dosage injectable form (e.g., solution, suspension, emulsion) in association with a pharmaceutically acceptable vehicle. Such vehicles are inherently nontoxic and non-therapeutic. Examples are water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Nonaqueous vehicles such as fixed oils, ethyl oleate, or liposome carriers may also be used. The vehicle may contain minor amounts of additives, such as buffers and preservatives, which enhance isotonicity and chemical stability or otherwise enhance therapeutic potential. The antibodies' concentrations in such vehicles are typically in the range of about 1 mg/ml to about 10 mg/ml. Immunotherapeutic methods are further described in the literature (U.S. Pat. No. 5,859,206; WO0073469).
Specific Biotherapeutics
In a preferred embodiment, a GPC-interacting protein may have biotherapeutic applications. Biotherapeutic agents formulated in pharmaceutically acceptable carriers and dosages may be used to activate or inhibit signal transduction pathways. This modulation may be accomplished by binding a ligand, thus inhibiting the activity of the pathway; or by binding a receptor, either to inhibit activation of, or to activate, the receptor. Alternatively, the biotherapeutic may itself be a ligand capable of activating or inhibiting a receptor. Biotherapeutic agents and methods of producing them are described in detail in U.S. Pat. No. 6,146,628.
GPC ligand(s), antibodies to the ligand(s) or the GPC itself may be used as biotherapeutics to modulate the activity of GPC in the IRRTK or p21 pathways.
Nucleic Acid Modulators
Other preferred GPC-modulating agents comprise nucleic acid molecules, such as antisense oligomers or double stranded RNA (dsRNA), which generally inhibit GPC activity. Preferred nucleic acid modulators interfere with the function of the GPC nucleic acid such as DNA replication, transcription, translocation of the GPC RNA to the site of protein translation, translation of protein from the GPC RNA, splicing of the GPC RNA to yield one or more mRNA species, or catalytic activity which may be engaged in or facilitated by the GPC RNA.
In one embodiment, the antisense oligomer is an oligonucleotide that is sufficiently complementary to a GPC mRNA to bind to and prevent translation, preferably by binding to the 5′ untranslated region. GPC-specific antisense oligonucleotides, preferably range from at least 6 to about 200 nucleotides. In some embodiments the oligonucleotide is preferably at least 10, 15, or 20 nucleotides in length. In other embodiments, the oligonucleotide is preferably less than 50, 40, or 30 nucleotides in length. The oligonucleotide can be DNA or RNA or a chimeric mixture or derivatives or modified versions thereof, single-stranded or double-stranded. The oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone. The oligonucleotide may include other appending groups such as peptides, agents that facilitate transport across the cell membrane, hybridization-triggered cleavage agents, and intercalating agents.
In another embodiment, the antisense oligomer is a phosphothioate morpholino oligomer (PMO). PMOs are assembled from four different morpholino subunits, each of which contain one of four genetic bases (A, C, G, or T) linked to a six-membered morpholine ring. Polymers of these subunits are joined by non-ionic phosphodiamidate intersubunit linkages. Details of how to make and use PMOs and other antisense oligomers are well known in the art (e.g. see WO99/18193; Probst J C, Antisense Oligodeoxynucleotide and Ribozyme Design, Methods. (2000) 22(3):271-281; Summerton J, and Weller D. 1997 Antisense Nucleic Acid Drug Dev.: 7:187-95; U.S. Pat. No. 5,235,033; and U.S. Pat. No. 5,378,841).
Alternative preferred GPC nucleic acid modulators are double-stranded RNA species mediating RNA interference (RNAi). RNAi is the process of sequence-specific, post-transcriptional gene silencing in animals and plants, initiated by double-stranded RNA (dsRNA) that is homologous in sequence to the silenced gene. Methods relating to the use of RNAi to silence genes in C. elegans, Drosophila, plants, and humans are known in the art (Fire A, et al., 1998 Nature 391:806-811; Fire, A. Trends Genet. 15, 358-363 (1999); Sharp, P. A. RNA interference 2001. Genes Dev. 15, 485-490 (2001); Hammond, S. M., et al., Nature Rev. Genet. 2, 110-1119 (2001); Tuschl, T. Chem. Biochem. 2, 239-245 (2001); Hamilton, A. et al., Science 286, 950-952 (1999); Hammond, S. M., et al., Nature 404, 293-296 (2000); Zamore, P. D., et al., Cell 101, 25-33 (2000); Bernstein, E., et al., Nature 409, 363-366 (2001); Elbashir, S. M., et al., Genes Dev. 15, 188-200 (2001); WO0129058; WO9932619; Elbashir S M, et al., 2001 Nature 411:494-498).
Nucleic acid modulators are commonly used as research reagents, diagnostics, and therapeutics. For example, antisense oligonucleotides, which are able to inhibit gene expression with exquisite specificity, are often used to elucidate the function of particular genes (see, for example, U.S. Pat. No. 6,165,790). Nucleic acid modulators are also used, for example, to distinguish between functions of various members of a biological pathway. For example, antisense oligomers have been employed as therapeutic moieties in the treatment of disease states in animals and man and have been demonstrated in numerous clinical trials to be safe and effective (Milligan J F, et al, Current Concepts in Antisense Drug Design, J Med Chem. (1993) 36:1923-1937; Tonkinson J L et al., Antisense Oligodeoxynucleotides as Clinical Therapeutic Agents, Cancer Invest. (1996) 14:54-65). Accordingly, in one aspect of the invention, a GPC-specific nucleic acid modulator is used in an assay to further elucidate the role of the GPC in the IRRTK or p21 pathways, and/or its relationship to other members of the pathway. In another aspect of the invention, a GPC-specific antisense oligomer is used as a therapeutic agent for treatment of IRRTK or p21-related disease states.
Assay Systems
The invention provides assay systems and screening methods for identifying specific modulators of GPC activity. As used herein, an “assay system” encompasses all the components required for performing and analyzing results of an assay that detects and/or measures a particular event. In general, primary assays are used to identify or confirm a modulator's specific biochemical or molecular effect with respect to the GPC nucleic acid or protein. In general, secondary assays further assess the activity of a GPC modulating agent identified by a primary assay and may confirm that the modulating agent affects GPC in a manner relevant to the IRRTK or p21 pathways. In some cases, GPC modulators will be directly tested in a secondary assay.
In a preferred embodiment, the screening method comprises contacting a suitable assay system comprising a GPC polypeptide or nucleic acid with a candidate agent under conditions whereby, but for the presence of the agent, the system provides a reference activity (e.g. binding activity), which is based on the particular molecular event the screening method detects. A statistically significant difference between the agent-biased activity and the reference activity indicates that the candidate agent modulates GPC activity, and hence the IRRTK or p21 pathways. The GPC polypeptide or nucleic acid used in the assay may comprise any of the nucleic acids or polypeptides described above.
Primary Assays
The type of modulator tested generally determines the type of primary assay.
Primary Assays for Small Molecule Modulators
For small molecule modulators, screening assays are used to identify candidate modulators. Screening assays may be cell-based or may use a cell-free system that recreates or retains the relevant biochemical reaction of the target protein (reviewed in Sittampalam G S et al., Curr Opin Chem Biol (1997) 1:384-91 and accompanying references). As used herein the term “cell-based” refers to assays using live cells, dead cells, or a particular cellular fraction, such as a membrane, endoplasmic reticulum, or mitochondrial fraction. The term “cell free” encompasses assays using substantially purified protein (either endogenous or recombinantly produced), partially purified or crude cellular extracts. Screening assays may detect a variety of molecular events, including protein-DNA interactions, protein-protein interactions (e.g., receptor-ligand binding), transcriptional activity (e.g., using a reporter gene), enzymatic activity (e.g., via a property of the substrate), activity of second messengers, immunogenicty and changes in cellular morphology or other cellular characteristics. Appropriate screening assays may use a wide range of detection methods including fluorescent, radioactive, calorimetric, spectrophotometric, and amperometric methods, to provide a read-out for the particular molecular event detected.
Cell-based screening assays usually require systems for recombinant expression of GPC and any auxiliary proteins demanded by the particular assay. Appropriate methods for generating recombinant proteins produce sufficient quantities of proteins that retain their relevant biological activities and are of sufficient purity to optimize activity and assure assay reproducibility. Yeast two-hybrid and variant screens, and mass spectrometry provide preferred methods for determining protein-protein interactions and elucidation of protein complexes. In certain applications, when GPC-interacting proteins are used in screens to identify small molecule modulators, the binding specificity of the interacting protein to the GPC protein may be assayed by various known methods such as substrate processing (e.g. ability of the candidate GPC-specific binding agents to function as negative effectors in GPC-expressing cells), binding equilibrium constants (usually at least about 10⁷M⁻¹, preferably at least about 10⁸M⁻¹, more preferably at least about 10⁹M⁻¹), and immunogenicity (e.g. ability to elicit GPC specific antibody in a heterologous host such as a mouse, rat, goat or rabbit). For enzymes and receptors, binding may be assayed by, respectively, substrate and ligand processing.
The screening assay may measure a candidate agent's ability to specifically bind to or modulate activity of a GPC polypeptide, a fusion protein thereof, or to cells or membranes bearing the polypeptide or fusion protein. The GPC polypeptide can be full length or a fragment thereof that retains functional GPC activity. The GPC polypeptide may be fused to another polypeptide, such as a peptide tag for detection or anchoring, or to another tag. The GPC polypeptide is preferably human GPC, or is an ortholog or derivative thereof as described above. In a preferred embodiment, the screening assay detects candidate agent-based modulation of GPC interaction with a binding target, such as an endogenous or exogenous protein or other substrate that has GPC-specific binding activity, and can be used to assess normal GPC gene function.
Suitable assay formats that may be adapted to screen for GPC modulators are known in the art. Preferred screening assays are high throughput or ultra high throughput and thus provide automated, cost-effective means of screening compound libraries for lead compounds (Fernandes P B, Curr Opin Chem Biol (1998) 2:597-603; Sundberg S A, Curr Opin Biotechnol 2000, 11:47-53). In one preferred embodiment, screening assays uses fluorescence technologies, including fluorescence polarization, time-resolved fluorescence, and fluorescence resonance energy transfer. These systems offer means to monitor protein-protein or DNA-protein interactions in which the intensity of the signal emitted from dye-labeled molecules depends upon their interactions with partner molecules (e.g., Selvin P R, Nat Struct Biol (2000) 7:730-4; Fernandes P B, supra; Hertzberg R P and Pope A J, Curr Opin Chem Biol (2000) 4:445-451).
A variety of suitable assay systems may be used to identify candidate GPC and IRRTK or p21 pathways modulators (e.g. U.S. Pat. Nos. 5,550,019 and 6,133,437 (apoptosis assays), among others). Specific preferred assays are described in more detail below.
Apoptosis assays. Assays for apoptosis may be performed by terminal deoxynucleotidyl transferase-mediated digoxigenin-11-dUTP nick end labeling (TUNEL) assay. The TUNEL assay is used to measure nuclear DNA fragmentation characteristic of apoptosis (Lazebnik et al., 1994, Nature 371, 346), by following the incorporation of fluorescein-dUTP (Yonehara et al., 1989, J. Exp. Med. 169, 1747). Apoptosis may further be assayed by acridine orange staining of tissue culture cells (Lucas, R., et al., 1998, Blood 15:4730-41). An apoptosis assay system may comprise a cell that expresses a GPC, and that optionally has defective IRRTK or p21 function (e.g. IRRTK or p21 is over-expressed or under-expressed relative to wild-type cells). A test agent can be added to the apoptosis assay system and changes in induction of apoptosis relative to controls where no test agent is added, identify candidate IRRTK or p21 modulating agents. In some embodiments of the invention, an apoptosis assay may be used as a secondary assay to test a candidate IRRTK or p21 modulating agents that is initially identified using a cell-free assay system. An apoptosis assay may also be used to test whether GPC function plays a direct role in apoptosis. For example, an apoptosis assay may be performed on cells that over- or under-express GPC relative to wild type cells. Differences in apoptotic response compared to wild type cells suggests that the GPC plays a direct role in the apoptotic response. Apoptosis assays are described further in U.S. Pat. No. 6,133,437.
Cell proliferation and cell cycle assays. Cell proliferation may be assayed via bromodeoxyuridine (BRDU) incorporation. This assay identifies a cell population undergoing DNA synthesis by incorporation of BRDU into newly-synthesized DNA. Newly-synthesized DNA may then be detected using an anti-BRDU antibody (Hoshino et al., 1986, Int. J. Cancer 38, 369; Campana et al., 1988, J. Immunol. Meth. 107, 79), or by other means.
Cell Proliferation may also be examined using [³H]-thymidine incorporation (Chen, J., 1996, Oncogene 13:1395-403; Jeoung, J., 1995, J. Biol. Chem. 270:18367-73). This assay allows for quantitative characterization of S-phase DNA syntheses. In this assay, cells synthesizing DNA will incorporate [³H]-thymidine into newly synthesized DNA. Incorporation can then be measured by standard techniques such as by counting of radioisotope in a scintillation counter (e.g., Beckman L S 3800 Liquid Scintillation Counter).
Cell proliferation may also be assayed by colony formation in soft agar (Sambrook et al., Molecular Cloning, Cold Spring Harbor (1989)). For example, cells transformed with GPC are seeded in soft agar plates, and colonies are measured and counted after two weeks incubation.
Involvement of a gene in the cell cycle may be assayed by flow cytometry (Gray J W et al. (1986) Int J Radiat Biol Relat Stud Phys Chem Med 49:237-55). Cells transfected with a GPC may be stained with propidium iodide and evaluated in a flow cytometer (available from Becton Dickinson).
Accordingly, a cell proliferation or cell cycle assay system may comprise a cell that expresses a GPC, and that optionally has defective IRRTK or p21 function (e.g. IRRTK or p21 is over-expressed or under-expressed relative to wild-type cells). A test agent can be added to the assay system and changes in cell proliferation or cell cycle relative to controls where no test agent is added, identify candidate IRRTK or p21 modulating agents. In some embodiments of the invention, the cell proliferation or cell cycle assay may be used as a secondary assay to test a candidate IRRTK or p21 modulating agents that is initially identified using another assay system such as a cell-free assay system. A cell proliferation assay may also be used to test whether GPC function plays a direct role in cell proliferation or cell cycle. For example, a cell proliferation or cell cycle assay may be performed on cells that over- or under-express GPC relative to wild type cells. Differences in proliferation or cell cycle compared to wild type cells suggests that the GPC plays a direct role in cell proliferation or cell cycle.
Angiogenesis. Angiogenesis may be assayed using various human endothelial cell systems, such as umbilical vein, coronary artery, or dermal cells. Suitable assays include Alamar Blue based assays (available from Biosource International) to measure proliferation; migration assays using fluorescent molecules, such as the use of Becton Dickinson Falcon HTS FluoroBlock cell culture inserts to measure migration of cells through membranes in presence or absence of angiogenesis enhancer or suppressors; and tubule formation assays based on the formation of tubular structures by endothelial cells on Matrigel® (Becton Dickinson). Accordingly, an angiogenesis assay system may comprise a cell that expresses a GPC, and that optionally has defective IRRTK or p21 function (e.g. IRRTK or p21 is over-expressed or under-expressed relative to wild-type cells). A test agent can be added to the angiogenesis assay system and changes in angiogenesis relative to controls where no test agent is added, identify candidate IRRTK or p21 modulating agents. In some embodiments of the invention, the angiogenesis assay may be used as a secondary assay to test a candidate IRRTK or p21 modulating agents that is initially identified using another assay system. An angiogenesis assay may also be used to test whether GPC function plays a direct role in cell proliferation. For example, an angiogenesis assay may be performed on cells that over- or under-express GPC relative to wild type cells. Differences in angiogenesis compared to wild type cells suggests that the GPC plays a direct role in angiogenesis.
Hypoxic induction. The alpha subunit of the transcription factor, hypoxia inducible factor-1 (HIF-1), is upregulated in tumor cells following exposure to hypoxia in vitro. Under hypoxic conditions, HIF-1 stimulates the expression of genes known to be important in tumour cell survival, such as those encoding glyolytic enzymes and VEGF. Induction of such genes by hypoxic conditions may be assayed by growing cells transfected with GPC in hypoxic conditions (such as with 0.1% O2, 5% CO2, and balance N2, generated in a Napco 7001 incubator (Precision Scientific)) and normoxic conditions, followed by assessment of gene activity or expression by Taqman®. For example, a hypoxic induction assay system may comprise a cell that expresses a GPC, and that optionally has a mutated IRRTK or p21 (e.g. IRRTK or p21 is over-expressed or under-expressed relative to wild-type cells). A test agent can be added to the hypoxic induction assay system and changes in hypoxic response relative to controls where no test agent is added, identify candidate IRRTK or p21 modulating agents. In some embodiments of the invention, the hypoxic induction assay may be used as a secondary assay to test a candidate IRRTK or p21 modulating agents that is initially identified using another assay system. A hypoxic induction assay may also be used to test whether GPC function plays a direct role in the hypoxic response. For example, a hypoxic induction assay may be performed on cells that over- or under-express GPC relative to wild type cells. Differences in hypoxic response compared to wild type cells suggests that the GPC plays a direct role in hypoxic induction.
Cell adhesion. Cell adhesion assays measure adhesion of cells to purified adhesion proteins, or adhesion of cells to each other, in presence or absence of candidate modulating agents. Cell-protein adhesion assays measure the ability of agents to modulate the adhesion of cells to purified proteins. For example, recombinant proteins are produced, diluted to 2.5 g/mL in PBS, and used to coat the wells of a microtiter plate. The wells used for negative control are not coated. Coated wells are then washed, blocked with 1% BSA, and washed again. Compounds are diluted to 2× final test concentration and added to the blocked, coated wells. Cells are then added to the wells, and the unbound cells are washed off. Retained cells are labeled directly on the plate by adding a membrane-permeable fluorescent dye, such as calcein-AM, and the signal is quantified in a fluorescent microplate reader.
Cell-cell adhesion assays measure the ability of agents to modulate binding of cell adhesion proteins with their native ligands. These assays use cells that naturally or recombinantly express the adhesion protein of choice. In an exemplary assay, cells expressing the cell adhesion protein are plated in wells of a multiwell plate. Cells expressing the ligand are labeled with a membrane-permeable fluorescent dye, such as BCECF, and allowed to adhere to the monolayers in the presence of candidate agents. Unbound cells are washed off, and bound cells are detected using a fluorescence plate reader.
High-throughput cell adhesion assays have also been described. In one such assay, small molecule ligands and peptides are bound to the surface of microscope slides using a microarray spotter, intact cells are then contacted with the slides, and unbound cells are washed off. In this assay, not only the binding specificity of the peptides and modulators against cell lines are determined, but also the functional cell signaling of attached cells using immunofluorescence techniques in situ on the microchip is measured (Falsey J R et al., Bioconjug Chem. May-June 2001;12(3):346-53).
Primary Assays for Antibody Modulators
For antibody modulators, appropriate primary assays test is a binding assay that tests the antibody's affinity to and specificity for the GPC protein. Methods for testing antibody affinity and specificity are well known in the art (Harlow and Lane, 1988, 1999, supra). The enzyme-linked immunosorbant assay (ELISA) is a preferred method for detecting GPC-specific antibodies; others include FACS assays, radioimmunoassays, and fluorescent assays.
Primary Assays for Nucleic Acid Modulators
For nucleic acid modulators, primary assays may test the ability of the nucleic acid modulator to inhibit or enhance GPC gene expression, preferably mRNA expression. In general, expression analysis comprises comparing GPC expression in like populations of cells (e.g., two pools of cells that endogenously or recombinantly express GPC) in the presence and absence of the nucleic acid modulator. Methods for analyzing mRNA and protein expression are well known in the art. For instance, Northern blotting, slot blotting, ribonuclease protection, quantitative RT-PCR (e.g., using the TaqMan®, PE Applied Biosystems), or microarray analysis may be used to confirm that GPC MRNA expression is reduced in cells treated with the nucleic acid modulator (e.g., Current Protocols in Molecular Biology (1994) Ausubel F M et al., eds., John Wiley & Sons, Inc., chapter 4; Freeman W M et al., Biotechniques (1999) 26:112-125; Kallioniemi O P, Ann Med 2001, 33:142-147; Blohm D H and Guiseppi-Elie, A Curr Opin Biotechnol 2001, 12:41-47). Protein expression may also be monitored. Proteins are most commonly detected with specific antibodies or antisera directed against either the GPC protein or specific peptides. A variety of means including Western blotting, ELISA, or in situ detection, are available (Harlow E and Lane D, 1988 and 1999, supra).
Secondary Assays
Secondary assays may be used to further assess the activity of GPC-modulating agent identified by any of the above methods to confirm that the modulating agent affects GPC in a manner relevant to the IRRTK or p21 pathways. As used herein, GPC-modulating agents encompass candidate clinical compounds or other agents derived from previously identified modulating agent. Secondary assays can also be used to test the activity of a modulating agent on a particular genetic or biochemical pathway or to test the specificity of the modulating agent's interaction with GPC.
Secondary assays generally compare like populations of cells or animals (e.g., two pools of cells or animals that endogenously or recombinantly express GPC) in the presence and absence of the candidate modulator. In general, such assays test whether treatment of cells or animals with a candidate GPC-modulating agent results in changes in the IRRTK or p21 pathways in comparison to untreated (or mock- or placebo-treated) cells or animals. Certain assays use “sensitized genetic backgrounds”, which, as used herein, describe cells or animals engineered for altered expression of genes in the IRRTK or p21 or interacting pathways.
Cell-Based Assays
Cell based assays may use cell lines known to have defective IRRTK or p21 function, such as HCT116 colon cancer cells with defective p21, available from American Type Culture Collection (ATCC), Manassas, Va.).
Cell based assays may detect endogenous IRRTK or p21 pathway activity or may rely on recombinant expression of IRRTK or p21 pathway components. Any of the aforementioned assays may be used in this cell-based format. Candidate modulators are typically added to the cell media but may also be injected into cells or delivered by any other efficacious means.
Animal Assays
A variety of non-human animal models of normal or defective IRRTK or p21 pathways may be used to test candidate GPC modulators. Models for defective IRRTK or p21 pathways typically use genetically modified animals that have been engineered to mis-express (e.g., over-express or lack expression in) genes involved in the IRRTK or p21 pathways. Assays generally require systemic delivery of the candidate modulators, such as by oral administration, injection, etc.
In a preferred embodiment, IRRTK or p21 pathways activity is assessed by monitoring neovascularization and angiogenesis. Animal models with defective and normal IRRTK or p21 are used to test the candidate modulator's affect on GPC in Matrigel® assays. Matrigel® is an extract of basement membrane proteins, and is composed primarily of laminin, collagen IV, and heparin sulfate proteoglycan. It is provided as a sterile liquid at 4° C., but rapidly forms a solid gel at 37° C. Liquid Matrigel® is mixed with various angiogenic agents, such as bFGF and VEGF, or with human tumor cells which over-express the GPC. The mixture is then injected subcutaneously(SC) into female athymic nude mice (Taconic, Germantown, N.Y.) to support an intense vascular response. Mice with Matrigel® pellets may be dosed via oral (PO), intraperitoneal (IP), or intravenous (IV) routes with the candidate modulator. Mice are euthanized 5-12 days post-injection, and the Matrigel® pellet is harvested for hemoglobin analysis (Sigma plasma hemoglobin kit). Hemoglobin content of the gel is found to correlate the degree of neovascularization in the gel.
In another preferred embodiment, the effect of the candidate modulator on GPC is assessed via tumorigenicity assays. In one example, xenograft human tumors are implanted SC into female athymic mice, 6-7 week old, as single cell suspensions either from a pre-existing tumor or from in vitro culture. The tumors which express the GPC endogenously are injected in the flank, 1×10⁵to 1×10⁷cells per mouse in a volume of 100 μL using a 27 gauge needle. Mice are then ear tagged and tumors are measured twice weekly. Candidate modulator treatment is initiated on the day the mean tumor weight reaches 100 mg. Candidate modulator is delivered IV, SC, IP, or PO by bolus administration. Depending upon the pharmacokinetics of each unique candidate modulator, dosing can be performed multiple times per day. The tumor weight is assessed by measuring perpendicular diameters with a caliper and calculated by multiplying the measurements of diameters in two dimensions. At the end of the experiment, the excised tumors maybe utilized for biomarker identification or further analyses. For immunohistochemistry staining, xenograft tumors are fixed in 4% paraformaldehyde, 0.1M phosphate, pH 7.2, for 6 hours at 4° C., immersed in 30% sucrose in PBS, and rapidly frozen in isopentane cooled with liquid nitrogen.
Diagnostic and Therapeutic Uses
Specific GPC-modulating agents are useful in a variety of diagnostic and therapeutic applications where disease or disease prognosis is related to defects in the IRRTK or p21 pathways, such as angiogenic, apoptotic, or cell proliferation disorders. Accordingly, the invention also provides methods for modulating the IRRTK or p21 pathways in a cell, preferably a cell pre-determined to have defective or impaired IRRTK or p21 function (e.g. due to overexpression, underexpression, or misexpression of IRRTK or p21, or due to gene mutations), comprising the step of administering an agent to the cell that specifically modulates GPC activity. Preferably, the modulating agent produces a detectable phenotypic change in the cell indicating that the IRRTK or p21 function is restored. The phrase “function is restored”, and equivalents, as used herein, means that the desired phenotype is achieved, or is brought closer to normal compared to untreated cells. For example, with restored IRRTK or p21 function, cell proliferation and/or progression through cell cycle may normalize, or be brought closer to normal relative to untreated cells. The invention also provides methods for treating disorders or disease associated with impaired IRRTK or p21 function by administering a therapeutically effective amount of a GPC-modulating agent that modulates the IRRTK or p21 pathways. The invention further provides methods for modulating GPC function in a cell, preferably a cell predetermined to have defective or impaired GPC function, by administering a GPC-modulating agent. Additionally, the invention provides a method for treating disorders or disease associated with impaired GPC function by administering a therapeutically effective amount of a GPC-modulating agent.
The discovery that GPC is implicated in IRRTK or p21 pathways provides for a variety of methods that can be employed for the diagnostic and prognostic evaluation of diseases and disorders involving defects in the IRRTK or p21 pathways and for the identification of subjects having a predisposition to such diseases and disorders.
Various expression analysis methods can be used to diagnose whether GPC expression occurs in a particular sample, including Northern blotting, slot blotting, ribonuclease protection, quantitative RT-PCR, and microarray analysis. (e.g., Current Protocols in Molecular Biology (1994) Ausubel F M et al., eds., John Wiley & Sons, Inc., chapter 4; Freeman W M et al., Biotechniques (1999) 26:112-125; Kallioniemi O P, Ann Med 2001, 33:142-147; Blohm and Guiseppi-Elie, Curr Opin Biotechnol 2001, 12:41-47). Tissues having a disease or disorder implicating defective IRRTK or p21 signaling that express a GPC, are identified as amenable to treatment with a GPC modulating agent. In a preferred application, the IRRTK or p21 defective tissue overexpresses a GPC relative to normal tissue. For example, a Northern blot analysis of mRNA from tumor and normal cell lines, or from tumor and matching normal tissue samples from the same patient, using full or partial GPC cDNA sequences as probes, can determine whether particular tumors express or overexpress GPC. Alternatively, the TaqMan® is used for quantitative RT-PCR analysis of GPC expression in cell lines, normal tissues and tumor samples (PE Applied Biosystems).
Various other diagnostic methods may be performed, for example, utilizing reagents such as the GPC oligonucleotides, and antibodies directed against a GPC, as described above for: (1) the detection of the presence of GPC gene mutations, or the detection of either over- or under-expression of GPC MRNA relative to the non-disorder state; (2) the detection of either an over- or an under-abundance of GPC gene product relative to the non-disorder state; and (3) the detection of perturbations or abnormalities in the signal transduction pathway mediated by GPC.
Thus, in a specific embodiment, the invention is drawn to a method for diagnosing a disease or disorder in a patient that is associated with alterations in GPC expression, the method comprising: a) obtaining a biological sample from the patient; b) contacting the sample with a probe for GPC expression; c) comparing results from step (b) with a control; and d) determining whether step (c) indicates a likelihood of the disease or disorder. Preferably, the disease is cancer, most preferably a cancer as shown in TABLE 1. The probe may be either DNA or protein, including an antibody.

EXAMPLES

The following experimental section and examples are offered by way of illustration and not by way of limitation.
I. Drosophila Screens
An EP overexpression screen was carried out in Drosophila to identify genes that modify the small eye phenotype resulting from expression of a dominant-negative InR in the eye. The EP collection contains a large set of Drosophila lines bearing P insertions that overexpress the gene in which they are inserted. Each EP line was crossed to lines expressing the dominant negative InR in the eye. Resulting progeny were examined for a change in the small eye phenotype. Sequence information surrounding the P insertion site was used to identify the overexpressed genes.
A dominant loss of function screen was carried out in Drosophila to identify genes that interact with the cyclin dependent kinase inhibitor, p21 (Bourne H R, et al., Nature (1990) 348(6297):125-132; Marshall C J, Trends Genet (1991) 7(3):91-95). Expression of the p21 gene from GMR-p21 transgene (Hay, B. A., et al. (1994) Development120:2121-2129) in the eye causes deterioration of normal eye morphology, resulting in reduced, rough eyes. Flies carrying this transgene were maintained as a stock (P 1025 F, genotype: y w; P{p21-pExp-gl-w[+]Hsp70(3′UTR)-5}). Females of this stock were crossed to a collection of males carrying piggyBac insertions (Fraser M et al., Virology (1985) 145:356-361). Resulting progeny carrying both the transgene and transposons were scored for the effect of the transposon on the eye phenotype, i.e. whether the transposon enhanced or suppressed (or had no effect) the eye phenotype. All data was recorded and all modifiers were retested with a repeat of the original cross, and the retests were scored at least twice. Modifiers of the eye phenotype were identified as members of the p21 pathway.
The Drosophila Dally gene (Genbank Identifier number 3023638), was identified as an enhancer of the small eye phenotype in both the IRRTK and p21 screens, and hence a member of the IRRTK and p21pathways. Human orthologs of the modifiers are referred to herein as GPC.
BLAST analysis (Altschul et al., supra) was employed to identify Targets from Drosophila modifiers. For example, representative sequences from GPC, GI#s 4504081, 4758462, 4504083, 4758464, and 5031719 (SEQ ID NOs:13, 17, 19, 20, and 22, respectively), share 24%, 24%, 22%, 22%, 28, and 23% amino acid identity, respectively, with the Drosophila Dally.
Various domains, signals, and functional subunits in proteins were analyzed using the PSORT (Nakai K., and Horton P., Trends Biochem Sci, 1999, 24:34-6; Kenta Nakai, Protein sorting signals and prediction of subcellular localization, Adv. Protein Chem. 54, 277-344 (2000)), PFAM (Bateman A., et al., Nucleic Acids Res, 1999, 27:260-2), SMART (Ponting C P, et al., SMART: identification and annotation of domains from signaling and extracellular protein sequences. Nucleic Acids Res. Jan. 1, 1999;27(1):229-32), TM-HMM (Erik L. L. Sonnhammer, Gunnar von Heijne, and Anders Krogh: A hidden Markov model for predicting transmembrane helices in protein sequences. In Proc. of Sixth Int. Conf. on Intelligent Systems for Molecular Biology, p 175-182 Ed J. Glasgow, T. Littlejohn, F. Major, R. Lathrop, D. Sankoff, and C. Sensen Menlo Park, Calif.: AAAI Press, 1998), and clust (Remm M, and Sonnhammer E. Classification of transmembrane protein families in the Caenorhabditis elegans genome and identification of human orthologs. Genome Res. November 2000; 10(11): 1679-89) programs. For example, the glypican domains (PFAM 01153) of GPC from GI#s4504081, 4758462, 4504083, 4758464, and 5031719 (SEQ ID NOs:13, 17, 19, 20, and 22, respectively) are located at approximately amino acid residues 2 to 557, 4 to 578, 1 to 555, 2 to 572, and 7 to 554, respectively
II. High-Throughput In Vitro Binding Assay.
³³P-labeled GPC peptide is added in an assay buffer (100 mM KCl, 20 mM HEPES pH 7.6, 1 mM MgCl₂, 1% glycerol, 0.5% NP-40, 50 mM beta-mercaptoethanol, 1 mg/ml BSA, cocktail of protease inhibitors) along with a test agent to the wells of a Neutralite-avidin coated assay plate and incubated at 25° C. for 1 hour. Biotinylated substrate is then added to each well and incubated for 1 hour. Reactions are stopped by washing with PBS, and counted in a scintillation counter. Test agents that cause a difference in activity relative to control without test agent are identified as candidate IRRTK or p21 modulating agents.
III. Immunoprecipitations and Immunoblotting
For coprecipitation of transfected proteins, 3×10⁶appropriate recombinant cells containing the GPC proteins are plated on 10-cm dishes and transfected on the following day with expression constructs. The total amount of DNA is kept constant in each transfection by adding empty vector. After 24 h, cells are collected, washed once with phosphate-buffered saline and lysed for 20 min on ice in 1 ml of lysis buffer containing 50 mM Hepes, pH 7.9, 250 mM NaCl, 20 mM-glycerophosphate, 1 mM sodium orthovanadate, 5 mM p-nitrophenyl phosphate, 2 mM dithiothreitol, protease inhibitors (complete, Roche Molecular Biochemicals), and 1% Nonidet P-40. Cellular debris is removed by centrifugation twice at 15,000×g for 15 min. The cell lysate is incubated with 25 μl of M2 beads (Sigma) for 2 h at 4° C. with gentle rocking.
After extensive washing with lysis buffer, proteins bound to the beads are solubilized by boiling in SDS sample buffer, fractionated by SDS-polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membrane and blotted with the indicated antibodies. The reactive bands are visualized with horseradish peroxidase coupled to the appropriate secondary antibodies and the enhanced chemiluminescence (ECL) Western blotting detection system (Amersham Pharmacia Biotech).
IV. Expression Analysis
All cell lines used in the following experiments are NCI (National Cancer Institute) lines, and are available from ATCC (American Type Culture Collection, Manassas, Va. 20110-2209). Normal and tumor tissues were obtained from Impath, UC Davis, Clontech, Stratagene, and Ambion.
TaqMan analysis was used to assess expression levels of the disclosed genes in various samples.
RNA was extracted from each tissue sample using Qiagen (Valencia, Calif.) RNeasy kits, following manufacturer's protocols, to a final concentration of 50 ng/μl. Single stranded cDNA was then synthesized by reverse transcribing the RNA samples using random hexamers and 500 ng of total RNA per reaction, following protocol 4304965 of Applied Biosystems (Foster City, Calif.).
Primers for expression analysis using TaqMan assay (Applied Biosystems, Foster City, Calif.) were prepared according to the TaqMan protocols, and the following criteria: a) primer pairs were designed to span introns to eliminate genomic contamination, and b) each primer pair produced only one product.
Taqman reactions were carried out following manufacturer's protocols, in 25 μl total volume for 96-well plates and 10 μl total volume for 384-well plates, using 300 nM primer and 250 nM probe, and approximately 25 ng of cDNA. The standard curve for result analysis was prepared using a universal pool of human cDNA samples, which is a mixture of cDNAs from a wide variety of tissues so that the chance that a target will be present in appreciable amounts is good. The raw data were normalized using 18S rRNA (universally expressed in all tissues and cells).
For each expression analysis, tumor tissue samples were compared with matched normal tissues from the same patient. A gene was considered overexpressed in a tumor when the level of expression of the gene was 2 fold or higher in the tumor compared with its matched normal sample. In cases where normal tissue was not available, a universal pool of cDNA samples was used instead. In these cases, a gene was considered overexpressed in a tumor sample when the difference of expression levels between a tumor sample and the average of all normal samples from the same tissue type was greater than 2 times the standard deviation of all normal samples (i.e., Tumor−average(all normal samples)>2×STDEV(all normal samples)).

Results are shown in Table 1. Data presented in bold indicate that greater than 50% of tested tumor samples of the tissue type indicated in row 1 exhibited over expression of the gene listed in column 1, relative to normal samples. Underlined data indicates that between 25% to 49% of tested tumor samples exhibited over expression. A modulator identified by an assay described herein can be further validated for therapeutic effect by administration to a tumor in which the gene is overexpressed. A decrease in tumor growth confirms therapeutic utility of the modulator. Prior to treating a patient with the modulator, the likelihood that the patient will respond to treatment can be diagnosed by obtaining a tumor sample from the patient, and assaying for expression of the gene targeted by the modulator. The expression data for the gene(s) can also be used as a diagnostic marker for disease progression. The assay can be performed by expression analysis as described above, by antibody directed to the gene target, or by any other available detection method.

TABLE 1


breast	.	colon	.	kidney	.	lung	.	ovary

GI#4504080	0	3	9	26	4	19	6	14	0	4
(SEQ ID NO: 1)
GI#5665747	0	3	6	26	8	19	3	14	2	4
(SEQ ID NO: 23)
GI#14763902	0	3	8	26	2	19	1	14	0	4
(SEQ ID NO: 24)
GI#13632295	1	3	8	26	4	19	1	14	1	4
(SEQ ID NO: 5)
GI#5360214	1	3	0	26	1	19	0	14	3	4
(SEQ ID NO: 6)
GI#4877642	1	3	9	26	6	19	3	14	0	4
(SEQ ID NO: 8)

Claims

1. A method of identifying a candidate IRRTK or p21 pathways modulating agent, said method comprising the steps of:

(a) providing an assay system comprising a purified GPC polypeptide or nucleic acid or a functionally active fragment or derivative thereof;

(b) contacting the assay system with a test agent under conditions whereby, but for the presence of the test agent, the system provides a reference activity; and

(c) detecting a test agent-biased activity of the assay system, wherein a difference between the test agent-biased activity and the reference activity identifies the test agent as a candidate IRRTK or p21 pathways modulating agent.

2. The method of claim 1 wherein the assay system comprises cultured cells that express the GPC polypeptide.

3. The method of claim 2 wherein the cultured cells additionally have defective IRRTK or p21 function.

4. The method of claim 1 wherein the assay system includes a screening assay comprising a GPC polypeptide, and the candidate test agent is a small molecule modulator.

5. The method of claim 4 wherein the assay is a binding assay.

6. The method of claim 1 wherein the assay system is selected from the group consisting of an apoptosis assay system, a cell proliferation assay system, an angiogenesis assay system, and a hypoxic induction assay system.

7. The method of claim 1 wherein the assay system includes a binding assay comprising a GPC polypeptide and the candidate test agent is an antibody.

8. The method of claim 1 wherein the assay system includes an expression assay comprising a GPC nucleic acid and the candidate test agent is a nucleic acid modulator.

9. The method of claim 8 wherein the nucleic acid modulator is an antisense oligomer.

10. The method of claim 8 wherein the nucleic acid modulator is a PMO.

11. The method of claim 1 additionally comprising:

(d) administering the candidate IRRTK or p21 pathways modulating agent identified in (c) to a model system comprising cells defective in IRRTK or p21 function and, detecting a phenotypic change in the model system that indicates that the IRRTK or p21 function is restored.

12. The method of claim 11 wherein the model system is a mouse model with defective IRRTK or p21 function.

13. A method for modulating a IRRTK or p21 pathways of a cell comprising contacting a cell defective in IRRTK or p21 function with a candidate modulator that specifically binds to a GPC polypeptide comprising an amino acid sequence selected from group consisting of SEQ ID NOs:13, 14, 15, 16, 17, 18, 19, 20, 21, and 22, whereby IRRTK or p21 function is restored.

14. The method of claim 13 wherein the candidate modulator is administered to a vertebrate animal predetermined to have a disease or disorder resulting from a defect in IRRTK or p21 function.

15. The method of claim 13 wherein the candidate modulator is selected from the group consisting of an antibody and a small molecule.

16. The method of claim 1, comprising the additional steps of:

(d) providing a secondary assay system comprising cultured cells or a non-human animal expressing GPC,

(e) contacting the secondary assay system with the test agent of (b) or an agent derived therefrom under conditions whereby, but for the presence of the test agent or agent derived therefrom, the system provides a reference activity; and

(f) detecting an agent-biased activity of the second assay system,

wherein a difference between the agent-biased activity and the reference activity of the second assay system confirms the test agent or agent derived therefrom as a candidate IRRTK or p21 pathways modulating agent,

and wherein the second assay detects an agent-biased change in the IRRTK or p21 pathways.

17. The method of claim 16 wherein the secondary assay system comprises cultured cells.

18. The method of claim 16 wherein the secondary assay system comprises a non-human animal.

19. The method of claim 18 wherein the non-human animal mis-expresses a IRRTK or p21 pathways gene.

20. A method of modulating IRRTK or p21 pathways in a mammalian cell comprising contacting the cell with an agent that specifically binds a GPC polypeptide or nucleic acid.

21. The method of claim 20 wherein the agent is administered to a mammalian animal predetermined to have a pathology associated with the IRRTK or p21 pathways.

22. The method of claim 20 wherein the agent is a small molecule modulator, a nucleic acid modulator, or an antibody.

23. A method for diagnosing a disease in a patient comprising:

(a) obtaining a biological sample from the patient;

(b) contacting the sample with a probe for GPC expression;

(c) comparing results from step (b) with a control;

(d) determining whether step (c) indicates a likelihood of disease.

24. The method of claim 23 wherein said disease is cancer.

25. The method according to claim 24, wherein said cancer is a cancer as shown in Table 1 as having >25% expression level.